Adaptação quantitativa do método oficial de análise de fosfatase alcalina em leite para queijos artesanais

Abstract

Milk pasteurization increases the microbiological safety of cheese. The safety of raw milk products is directly related to good milking and production practices. Artisanal cheeses are typically produced on a small scale, using mainly raw milk. In the last two decades, the 60-day aging rule, previously required for raw milk cheese commercialization as a way of guaranteeing its safety, has been exempted under legislation. In this context, alkaline phosphatase (ALP) is an indigenous raw milk enzyme used as a pasteurization marker both nationally and internationally. However, ALP analysis in cheese is not commonly performed in Brazil, and in other countries it relies on high-cost methods, limiting broader implementation. Therefore, this research aimed to propose a novel analytical approach for the quantification of ALP in artisanal cheeses. Artisanal cheeses made from raw and pasteurized milk were aged for 70 days and evaluated for ALP activity each 10 days. Three methods of enzyme extraction were used, including two methods based on organic solvent extraction, and a green method, based on water extraction. ALP was assessed from the extracts, using the official national method for milk research, and the resulting color was subjected to spectrophotometric analysis, reference quantitative approach for colorimetric assays, and instrumental color analysis. The results showed that the ALP activity decreased by approximately 50% throughout the 70 days of ripening and that the extraction method adopted significantly affected enzyme activity quantified (p < 0.05), with higher values for organic solvent extractions. However, although water extraction and enzyme activities estimated by b* showed detection and quantification limits above 10 mU/g, the international reference value for pasteurized milk cheeses, it was possible to distinguish clearly between raw and pasteurized milk cheeses. Additionally, it eliminates the need of color dilution, required for spectrophotometry analysis, and when compared to the fluorimetric reference method offers lower costs. Color coordinate b* was directly correlated with ALP activity, making it a suitable pasteurization marker. This could be a first step towards more assertive control of the thermal treatment of milk used in artisanal cheeses manufacturing by inspection agencies, in order to guarantee their microbiological safety and compliance with specific identity and quality regulations.